1. Field of the Invention
The present invention relates to a DNA encoding a polypeptide possessing endo-.beta.-N-acetylglucosaminidase A activity, and to a method for producing a polypeptide possessing endo-.beta.-N-acetylglucosaminidase A activity by the use of the DNA.
2. Discussion of the Related Art
In recent years, various physiological functions of the sugar chain moieties of molecules known as complex carbohydrates, such as glycoproteins and glycolipids, have drawn attention. At present, carbohydrate-decomposing enzymes serve as very useful tools for elucidation of the structure and biological activity of sugar chains. Endo-.beta.-N-acetylglucosaminidase, in particular, catalyzes the reaction in which the GlcNAc.beta.1-4GlcNAc bond of di-N-acetylchitobiose at the reduction end of the N-linked sugar chain of glycoproteins is broken to cut off the sugar chain from the protein and leave N-acetylglucosamine on the protein side. Endo-.beta.-N-acetylglucosaminidase has further been used for structural or functional analysis of glycoproteins.
In addition, some forms of endo-.beta.-N-acetylglucosaminidase are known to catalyze sugar chain rearrangement reactions; endo-.beta.-N-acetylglucosaminidase A from the Arthrobacter protoformiae AKU 0647 strain (hereinafter also referred to as Endo-A), in particular, has been reported to possess very potent sugar chain rearrangement activity (Japanese Patent Laid-Open No. 5-64594). Specifically, Endo-A efficiently catalyzes the reaction in which the N-binding oligomannose type sugar chain of glycoproteins is cut out and transferred to an acceptor carbohydrate or complex carbohydrate. The Endo-A enzyme is therefore very useful not only for the structural analysis of sugar chains of glycoproteins but also for other purposes such as modification of sugar chains of complex carbohydrates, and preparation of neoglycoproteins.
A known form of Endo-A is derived from Arthrobacter protoformiae [Applied and Environmental Microbiology, 55, 3107-3112 (1989)].
However, in the method in which Arthrobacter protoformiae is cultured to obtain Endo-A, proteases and other glycosidases are also produced. It has been difficult to separate and purify these co-present enzymes from Endo-A. Also, to induce Endo-A enzyme production, ovalbumin or a sugar peptide thereof must be added to the culture medium. There has therefore been a need for the development of a method enabling the production of highly pure Endo-A at low cost.
Although purification of Endo-A from Arthrobacter protoformiae is already known [Applied and Environmental Microbiology, 55, 3107-3112 (1989)], there has been no knowledge regarding the amino acid sequence or gene structure of Endo-A, and hence there is no method of Endo-A production by gene engineering.